The long-term goal of this project is to understand the molecular mechanism by which DNA mismatch repair (MMR) maintains to genomic stability. MMR plays an important role in replication fidelity by correcting mispairs generated during DNA replication. Mismatch recognition protein hMutS?, a heterodimer consisting of hMSH2 and hMSH6 subunits, is arguably the most important component in MMR. The hMSH6 subunit contains a PCNA interaction protein motif (called PIP box) and a PWWP domain at its N-terminus. While no clear function has been assigned to the PWWP domain, the PIP box was previously thought to be involved in recruiting hMutS? to mismatched DNA. Interestingly, recent studies have shown that depletion of the PIP box only moderately increases mutation frequencies, and does not affect hMutS? chromatin localization. Surprisingly, we show recently that the MSH6 PWWP domain physically interacts with histone mark H3K36me3 and is responsible for localizing hMutS? to replicating chromatin. These observations suggest that the PCNA-hMSH6 interaction is not for hMutS? recruitment and that the epigenetic H3K36me3 histone mark plays a critical role in MMR and genomic stability. Given the abundance of H3K36me3 in S phase and that PCNA interacts with both hMutS? and replicative DNA polymerases ? and ?, we hypothesize that the H3K36me3-hMutS? interaction in individual nucleosomes determines mutation rates in the corresponding DNA sequences, and that the PCNA-hMutS? interaction coordinates the DNA replication and MMR reactions at the replication fork. To test these hypotheses, three specific aims are proposed. Specific aim 1 is to determine genome-wide distributions of and interactions between H3K36me3 and hMutS? by ChIP- Seq analysis. Specific aim 2 is to study molecular details as to how the PCNA-hMutS? interaction regulates DNA synthesis and MMR. Specific aim 3 is to determine the impact of hMutS? interactions with H3K36me3 and PCNA on gene mutations using Exome-Sequencing analysis and a mutagenesis assay, respectively. A successful completion of the proposed study will not only elucidate novel mechanisms by which MMR maintains genome stability, but also provide potentially new biomarkers for cancer detection and therapy.